Project Summary/Abstract While it has long been recognized that the wide array of polymorphisms in major histocompatibility complex (MHC) in combination with the direct role of MHC in activation of T cells through the T cell receptor (TCR) makes MHC the primary antigenic barrier to transplantation, the molecular interactions between TCR and allogeneic MHC defining alloreactivity remain enigmatic. Alloreactivity is important both in fundamental understanding of T cell recognition of antigen, as well as in clinical allogeneic transplantation. The applicant hypothesizes that the repertoire of alloreactive T cells mediating graft vs. host disease (GvHD), a highly clinically-relevant manifestation of alloreactivity, is due to a combination of germ-line encoded affinity between a TCR and an MHC molecule, polyspecificity in the recognition of MHC bound peptides, and dual TCR expressing T cells. There are three specific aims to address this hypothesis: In Aim 1, the applicant proposes research that continues investigation of the influence of the TCR repertoire in GvHD;proposing to translate preliminary results from a mouse model demonstrating the participation of highly alloreactive dual TCR CD4 and CD8 T cells in GvHD to humans, including clinical feasibility studies for diagnostic test development. In Aim 2, the applicant proposes using a large scale TCR sequencing approach to generate a comprehensive alloreactive CD4 and CD8 T cell library and use it to examine the breadth of the repertoire of T cells mediating GvHD and identify molecular determinants of the TCR repertoire predisposing alloreactivity. In Aim 3, a direct comparison will be made between the recognition of a TCR with known conventional and allogeneic ligands, which will permit the elucidation of the relative flexibility in molecular interactions defining alloreactivity. Together, these studies will improve understanding of molecular interactions defining the alloreactive T cell response responsible for GvHD, enabling further investigation and rational design of improved diagnostics, immunosuppressive agents, and donor matching strategies.